Cancer is a leading cause of death and accounts for approximately 13% of all deaths in the world. Most cancers form solid tumors in tissues like head and neck, colon, breast, lung, liver and stomach, and are often characterized by low oxygen concentrations (hypoxia) and acidification of the microenvironment surrounding the tumor cells. Hypoxia and acidification of the extratumoral environment are both associated with aggressive tumor growth, metastasis formation and poor response to radiotherapy, surgery and/or to anticancer chemotherapy. The most important pathway that acts on changes in oxygen concentration is the ‘Hypoxia Inducible Factor-1 pathway’ (HIF-1 pathway). Under hypoxic conditions, the transcription factor HIF-1α is stabilized and binds to HIF-1β. The formed complex can translocate to the nucleus and bind to the hypoxic-responsive elements (HRE's) of genes involved in anaerobic metabolism, pH regulation, angiogenesis, cell proliferation and survival.
Carbonic anhydrases (CAs) form a large family of ubiquitous zinc metalloenzymes of great physiological importance. As catalysts of reversible hydration of carbon dioxide to bicarbonate and protons (CO2+H2OH++HCO3−), these enzymes participate in a variety of biological processes, including respiration, calcification, acid-base balance, bone resorption, formation of aqueous humor. To date, 16 isozymes are characterized from which 15 present in humans. CAs in humans are present in several tissues (e.g. GI tract, reproductive tract, skin, kidneys, lungs, eyes, . . . ) and are localized in different parts of the cell. Basically, there are several cytosolic forms (CA I-III, CA VII), four membrane-bound isozymes (CA IV, CA IX, CA XII and CA XIV), one mitochondrial form (CA V) as well as a secreted CA isozyme, CA VI.
It has been shown that some tumor cells predominantly express only some membrane-associated CA isozymes, such as CA IX and CA XII. CAs show considerable diversity in their tissue distribution, levels, and putative or established biological functions. Some of the CAs are expressed in almost all tissues (CA II), whereas the expression of others appears to be more restricted (e.g., CA VI and CA VII in salivary glands).
Furthermore, it is conceivable that CA activity might also be well exploited in tumors, since tumors often display a reversed pH gradient across the plasma membrane when compared with normal tissues, indicating the contribution of CAs in providing protons for acidification of the extracellular environment and bicarbonate ions to maintain a neutral intracellular milieu. In addition to the acidifying effect on the extracellular pH, it can influence the uptake of anticancer drugs and modulate the response of tumor cells to conventional therapy, such as radiation therapy. One of the CA isozymes, CA IX, shows restricted expression in normal tissues, but is tightly associated with different types of tumors. CA IX, originally detected in human carcinoma HeLa cells as a cell density-regulated antigen (Pastorekova et al, 1992), is strongly induced by tumor hypoxia, through a transcriptional activation by the HIF-1 pathway. Strong association between carbonic anhydrase CA IX expression and intratumoral hypoxia has been demonstrated in carcinomas. CA IX distribution is often examined in relation to the extent of necrosis as an indicator of severe hypoxia and to microvascular density as a measure of angiogenesis. Furthermore, CA IX associated with worse relapse-free survival and overall survival in patients with invasive tumors. CA IX is also a significant prognostic indicator of overall survival and metastasis-free survival after radiotherapy and chemoradiotherapy.
Hypoxia is linked with acidification of extracellular environment that facilitates tumor invasion and CA IX is believed to play a role in this process via its catalytic activity (Svastova et al, 2004). CA IX has a very high catalytic activity with the highest proton transfer rate among the known CAs, has been shown to acidify the extracellular environment and is therefore an interesting target for anticancer therapy, preferably in combination with conventional treatment schedules. Targeting CA IX would be preferred above targeting HIF-1, the master regulator of the transcriptional response of mammalian cells to oxygen deprivation, since controversial results have been reported depending on the cell type used, the subunit targeted, the site of tumor and the timing of HIF-1 inhibition (early or later in tumor growth). Furthermore, because most of the small-molecules inhibitors of HIF-1 affect multiple signalling pathways and/or targets indirectly associated with HIF, assessment of their activity as HIF inhibitors cannot be based on therapeutic efficacy, which might be unrelated to HIF inhibition (Melillo, 2006).
Recently, it has emerged that carbonic anhydrase inhibitors (CAIs) could have potential, besides the established role as diuretics and anti-glaucoma drugs, as novel anti-obesity, anti-cancer and anti-infective drugs. There are 2 main classes of carbonic anhydrase inhibitors: the metal complexing anions and the unsubstituted sulfonamides and their derivatives, which bind to the Zinc ion of the enzyme either by substituting the nonprotein zinc ligand or add to the metal coordination sphere (Supuran, 2008). However, the critical problem in designing these inhibitors is the high number of isozymes, the diffuse localization in tissues and the lack of isozyme selectivity of the presently available inhibitors.
All six classical CAIs (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, and dichlorophenamide) used in clinical medicine or as diagnostic tools, show some tumor growth inhibitory properties. Most of the clinically used sulfonamides mentioned above are systemically acting inhibitors showing several undesired side effects due to inhibition of many of the different CA isozymes present in the target tissue/organ (15 isoforms are presently known in humans). Therefore, many attempts to design and synthesize new sulfonamides were recently reported, in order to avoid such side effects. Isozymes associated to cell membranes (CA IV, CA IX, CA XII and CA XIV), with the enzyme active site generally oriented extracellularly, provide a rational basis for targeting. CA IX and CA XII are both extracellularly located on hypoxic tumor cells and are therefore the best candidates.
The ideal characteristics for specific CA IX inhibitors should demonstrate a relatively low inhibition constant (Ki in the nanomolar range) and should be relatively specific over the cytosolic enzymes CA I and CA II. A number of aromatic sulfonamides has been presented (see e.g. WO02004048544) that specifically bind to the extracellular components of the in particular CAIX enzyme, which show a higher specificity than those hitherto known in the art. Therapeutic and diagnostic sulfonamide agents are described in WO2006137092. In WO2008071421 it was shown that the inhibitory effect of heterocyclic sulfonamides can be further increased by oxidative substituents, in particular nitrosated or nitrosylated substituents, since such groups may increase the acidity of the zinc binding groups and as such being beneficial for the carbonic anhydrase inhibitory properties. Sulfonamide-based metal chelate complexes for imaging are described in WO2009089383. However, a large variation is reported in CA IX inhibitory constants for the sulfonamides as well as variation in the selectivity of the inhibitors. Sulfamate and sulfamide inhibitors have also been proposed as candidates (Winum et al, 2009).
Traditional anticancer therapy like surgery, irradiation and chemotherapy are used to treat cancer patients as a combined or single treatment. The basic principle of irradiation is to damage the cancer cells to such an extent that they will die. Free radicals are formed and damage the DNA immediately or they react with oxygen, creating reactive oxygen species which damage the cell and more specific the DNA in the cell. However when no or little oxygen is present, what is the case in hypoxic tumors, less reactive oxygen species are formed and the irradiation is not as effective. It has been shown that a 3 fold higher radiation dose is required to kill the same amount of hypoxic cells as compared under normal oxygen concentrations. The concept of radiosensitization of hypoxic cells emerged when certain compounds were able to mimic oxygen and thus enhance radiation damage. The first compounds which demonstrated radiosensitization were nitrobenzenes, followed by nitrofurans and 2-nitroimidazoles, such as misonidazole (see e.g. WO02006102759). Although in experimental tumor models enhanced radiation damage was observed, most of the clinical trials using misonidazole were unable to demonstrate a significant improvement in radiation response, although benefit was seen in certain subgroups of patients (Overgaard, 1989). The most likely explanation is the fact that the misonidazole doses were too low, limited by the risk of neurotoxicity. Alternative, better radiosensitizing drugs, such as etanidazole and pimonidazole, were synthesized and tested, but clinical results did not result in a significant therapeutic benefit. Less toxic drugs, such as nimorazole, could theoretically achieve lower sensitizing ability compared with misonidazole, but due to it far lower toxicity, much higher, clinically relevant doses can be obtained. Only clinical studies in patients with supraglottic and pharyngeal carcinomas (DAHANCA 5) resulted in highly significant benefit in terms of improved loco-regional tumor control and disease-free survival (Overgaard et al, 1998). More specific targeting towards the hypoxic tumor cell using lower doses is therefore an important requisite for new compounds.
It now has surprisingly been found that the compounds of the invention, as represented by formula (1):
in which Z is Z1 represented by formula (2a):
                or Z is Z2 represented by formula (2b): (CH2)nCH2X        or Z is Z3 represented by formula (2c):        
R1 and R2 can be, each independently, H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, cyano or halogen atom,R3, R4, R6 and R7 can be, each independently H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, halogen atom, cyano, alkoxy, sulfonamide, sulfamate or sulfamide,R5 can be sulfonamide, sulfamate or sulfamide,X=sulfonamide, sulfamate or sulfamide,Y=O or S,and n=0, 1, 2, 3, 4 or 5, demonstrate not only a higher specificity for CA IX than any compound known in the art, but also have a much increased radiosensitizing effect. This is the more unexpected as the stereochemical orientation of the various active groups in the compounds of the invention is quite different from that of the compounds known in the art be it sulfonamides or nitroimidazoles. In other words, it is surprising that the radiosensitizing activity of the nitroimidazoles has been retained although the group now is part of a larger entity and the compounds are less neurotoxic.
Therefore the compounds of the invention have a significantly improved overall profile for treating solid tumours, such as tumours of the breast, brain, kidney, colorectal, lung, head and neck, bladder etc. compared to carbonic anhydrase inhibitors known in the art. Also other therapeutic fields such as treating eye disorders in particular, glaucoma, ocular hypertension, age-related macular degeneration, diabetic macular edema, diabetic retinopathy, hypertensive retinopathy and retinal vasculopathies, epilepsy, high-altitude disorders and neuromuscular diseases fall within the range of applications of the compounds of the invention.
Another finding is that the compounds of the invention also show an unexpected positive effect on radiosensitivity. Extracellular acidosis has been thought to be the result of excess production of lactic acid. However, glycolytic deficient cells (cells in which lactic acid production is hampered) result in tumors with a similar extent of extracellular acidosis, indicating other involved players aside lactic acid. Several studies have been shown that extracellular acidosis makes tumours less sensitive to irradiation treatment (Brizel et al, 2001; Quennet et al, 2006). Carbonic anhydrase inhibiting sulfonamides are able to reduce the extracellular acidosis in tumors and are therefore a possible tool to improve the sensitivity to irradiation of tumours. In addition, the fact that CA IX expression is limited in normal healthy tissue, while highly overexpressed in tumours, makes the carbonic anhydrase isozyme IX an attractive target within the concept.
On the other hand, hypoxic conditions in tumours make them less sensitive to the ionising radiation commonly used in radiotherapy (Thomlinson & Gray, 1955). Attracting the CA inhibitory compounds towards hypoxic cells, would greatly increase the possible therapeutic effect. This can be done using nitroimidazoles which are trapped in hypoxic cells after a two-fold electron reduction upon low oxygen conditions.
In other words, on the one hand there is a need to increase the anti-acidic, antitumorigenic effects and specificity of CA IX inhibiting sulf(on)amides and on the other hand there is a need to target specifically hypoxic cells using substituted nitroimidazoles to make compounds more suitable for radiosensitizing therapy. These needs are met by the present invention which provides multifunctional dual CAIX targeting drug compounds and preparations for the treatment of cancer in a patient in need thereof comprising compounds of formula 1a-c above.
Further objects of the present invention are also pharmaceutical compositions containing at least a compound of the present invention of formula (1a-c) together with non toxic adjuvants and/or carriers usually employed in the pharmaceutical field.
FIG. 1 shows Scheme 1.
Reagents and conditions: (i) 1 equiv. of 2-nitroimidazole, 1 equiv. of tert-butyl bromoacetate, 4 equiv. of potassium carbonate, MeCN, RT, 1 night: (ii) cocktail of trifluoroacetic acid/water/thioanisole 95/2.5/2.5 v/v, room temperature, 1 night; (iii) 1 equiv. of 4-dimethylaminopyridine (DMAP), 1 equiv. of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N,N-dimethylacetamide (DMA), room temperature, 2 days.
FIG. 2 shows scheme 2:
Reagents and conditions: (i) 1 equiv. of 1-(2-aminoethyl)-2-methyl-5-nitroimidazole dihydrochloride monohydrate, 1 equiv. SCN-Ph-SO2NH2, 2 equiv. of triethylamine, MeCN, room temperature, 1 hour.
FIG. 3 shows the scheme of the preferred compound of the invention
Reagents and conditions: (i) 1 equiv. of 1-(2-aminoethyl)-2-methyl-5-nitroimidazole dihydrochloride monohydrate, 4 equiv. of triethylamine, 1 equiv. of chlorosulfonylisocyanate, 1 equiv. of tert-butanol, CH2Cl2, rt, 1 hour; (ii) trifluoroacetic acid/CH2Cl2 7/3, rt, 6 hours.
FIG. 4 shows the scheme of the second preferred compound of the invention
Reagents and conditions: (i) 1 equiv. of 2-methyl-5-nitro-1-imidazolylethanol, N,N-dimethylacetamide, 3 equiv. sulfamoyl chloride, rt, 1 night.
The compounds of the present invention can be synthesised according to the following procedures. All reagents and solvents were of commercial quality and used without further purification, unless otherwise specified. All reactions were carried out under an inert atmosphere of nitrogen. TLC analyses were performed on silica gel 60 F254 plates (Merck Art. 1.05554). Spots were visualized under 254 nm UV illumination, or by ninhydrin solution spraying. Melting points were determined on a Büchi Melting Point 510 and are uncorrected. 1H and 13C NMR spectra were recorded on Bruker DRX-400 spectrometer using DMSO-d6 as solvent and tetramethylsilane as internal standard. For 1H NMR spectra, chemical shifts are expressed in δ (ppm) downfield from tetramethylsilane, and coupling constants (J) are expressed in Hertz. Electron Ionization mass spectra were recorded in positive or negative mode on a Water MicroMass ZQ.
It is referred to the attached FIGS. 1 and 2.